We have used a murine in vivo infection selection model to isolate a hypervirulent C. trachomatis human strain. The virulent isolate produces infection and disease in the mouse female genital tract with similar pathology to that of human infection. These findings are the first description of a human strain that is virulent for the mouse thereby providing a much needed small animal model for the study of human infection and disease. Remarkably, comparative genomic studies of virulent and avirulent clonal strains revealed a mutation in only a single gene unambiguously identifying the gene (CT153) as a critical in vivo virulence factor. These findings will significantly impact our understanding of the pathogenesis and pathophysiology human infections. Moreover, these findings will provide new approaches to small animal modeling systems using human isolates that should advance future efforts in chlamydial vaccine design and development. Chlamydia trachomatis is the etiological agent of trachoma, the leading cause of preventable blindness. Trachoma presents distinct clinical syndromes ranging from mild and self-limiting to severe inflammatory disease. The underlying host and pathogen factors responsible for these diverse clinical outcomes are unclear. There is no available small animal model that mimics human infection disease. We have isolated a hypervirulent human strain that is highly infectious for the mouse that causes infection and disease very similar to that characteristic of human infection. By comparative genomic analysis we found that virulent and avirulent isogenic isolates have mutations in only a single gene (CT135);strongly implicating this gene in chlamydial virulence and pathogenesis. These findings have significant implications for human genotyping studies to identify individuals at greater risk of post-infection complications and alternate therapeutic management. Future studies will focus on understanding the function of CT135. Defining its role in pathogenesis could provide new insights important to vaccine design and development.